Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSconsequences to EG-2-GFP trafficking of blocks to secretion imposed by pharmacological or mutational insults. Our initial results indicate that EG-2-GFPshows localization to the ER and is mostly absent from the Spitzenkorper, suggesting trafficking through a classical ER to Golgi secretory pathway andterminal secretion along lateral hypahl walls. Additonally, targeted blocks to the secretory pathway indicate a potential role of endosomes in EG-2-GFPtrafficking.193. Towards understanding the endoplasmic reticulum associated degradation process of misfolded glycoproteins in Neurospora crassa. GeorgiosTzelepis 1 , Hiroto Hirayama 2 , Tadashi Suzuki 2 , Akira Hosomi 2 , Mukesh Dubey 1 , Magnus Karlsson 1 . 1) Uppsala BioCenter, Department of Forest Mycology andPlant Pathology, Swedish University of Agricultural Sciences, Box 7026, 75007, Uppsala, Sweden; 2) Glycometabolome Team, Systems GlycobiologyResearch Group, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako Saitama 351-0198, Japan.N-glycosylation is an important post-translational modification of proteins, which occurs in the Endoplasmic Reticulum (ER). These N-linked glycans arereported to play an important role in correct protein folding. Glycoproteins that are unable to fold properly are subjected to destruction by an ERassociateddegradation process (ERAD). Degradation of these glycoproteins generates free oligosaccharides (fOs). In animal and plant cells mainly threetypes of hydrolytic enzymes are involved in the ERAD pathway. First, PNGases which cleave the sugar chain from the protein releasing fOs with N,N'-diacetylchitobiose moieties (fOs-GN2). Secondly, ENGases which catalyse the glycosidic bonds in N,N'-diacetylchitobiose moieties, generating fOs with asingle N-acetylglucoseamine at their reducing ends (fOs-GN1), and thirdly, a-mannosidases responsible for trimming the mannose chains before finaldegradation in lysosomes. The existence of this pathway in filamentous ascomycetes is unknown. In this study we investigate the function of ENGases in N.crassa by analysing the phenotype of deletion strain Dgh18-10 and quantifying the content and type of fOs (fOs-GN1 or fOs-GN2), using dual gradient highperformance liquid chromatography. Since cytosolic PNGase is enzymatically inactive in N. crassa, ENGases possibly have a crucial role in the ERADpathway. We found that deletion of an intracellular ENGase results in severe phenotypic effects. This deletion strain shows significantly slower growth ratein carbon-rich media but grows faster in abiotic stress conditions, indicating a more resistant cell wall. Moreover, the conidiation rate is higher in Dgh18-10compared to WT. Sexual reproduction is also affected, since no ascospores were observed in Dgh18-10. Additionally, the total amount of extracellularproteins was significantly lower in this deletion strain compared to WT. Finally, this mutation causes repression of three chitin synthase genes in N. crassa.Similar results were also observed in the mycoparasitic ascomycete Trichoderma atroviride. These data may suggest that deletion of cytosolic ENGaseleads to accumulation of misfolded glycoproteins in the fungal cytosol, which somehow affects its protein secretion/structure of cell wall. This is the firststudy of the ERAD pathway in filamentous ascomycetes.194. Saccharomyces cerevisiae spore development and protection against reactive oxygen species. Steve Gorsich, Tricia Stokes, Michelle Steidemann,Kyle Kern. Dept Biol, Central Michigan Univ, Mt Pleasant, MI.The generation of spores in S. cerevisiae is essential for sexual reproduction and survival of the organism. When diploid S. cerevisiae cells undergomeiotic division to produce four spores it is important for each spore to not only get a haploid copy of nuclear chromosomes, but also a completecomplement of organelles and potentially RNP granules. For instance mitochondria undergo temporally regulated fusion and fission events to assure thatmitochondria are represented equally in each of the resulting spores. When this network is not maintained, due to mutations in mitochondrial fissiongenes (e.g. dnm1/dnm1), it has been shown that fewer spores survive and the ones that do survive have reduced respiratory fitness. In addition tomutations affecting spore production we hypothesized that environmental factors could also influence spore development. In the present study, wedemonstrated that hydrogen peroxide can phenocopy the mitochondrial fission mutant’s phenotypes. Wild-type S. cerevisiae exposed to hydrogenperoxide have mitochondrial morphology and distribution defects during spore development, reduced spore viability, and decreased respiratorycompetency just as seen in dnm1/dnm1 fission mutants. We next hypothesized that the phenotypes associated with dnm1/dnm1 mitochondrial fissionmutants were caused by increased sensitivity to reactive oxygen species (ROS). To support this we demonstrated that dnm1/dnm1 mutants have anincrease in ROS during spore development. In addition, sporulation defects associated with dnm1/dnm1 or wild-type cells exposed to hydrogen peroxidewere rescued when we overexpressed oxidative stress protection genes. These findings suggest that the ability of S. cerevisiae to produce optimalnumbers of fit spores is heavily influenced by their ability to protect themselves from exogenous or endogenous ROS.195. Genetic analysis of the role of peroxisomes in the virulence and survival in Fusarium graminearum. K. Min 1 , H. Son 1 , J. Lee 2 , G. J. Choi 3 , J.-C. Kim 3 , Y.-W. Lee 1 . 1) Department of Agricultural Biotechnology and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921, Republic of Korea; 2)Department of Applied Biology, Dong-A University, Busan 604-714, Republic of Korea; 3) Eco-friendly New Materials Research Group, Research Center forBiobased Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, Daejeon 305-343, Republic of Korea.Peroxisomes are single-membrane-bound organelles that are required for diverse biochemical processes, including b-oxidation of fatty acids anddetoxification of reactive oxygen species (ROS). In this study, the role of peroxisomes was examined in Fusarium graminearum by functional analysis ofthree genes (PEX5, PEX6, and PEX7) encoding peroxin proteins required for peroxisomal protein import. PEX5 and PEX7 deletion mutants failed to localizethe fluorescently-tagged peroxisomal targeting signal type 1 (PTS1)- and PTS2-containing proteins to peroxisomes, respectively, whereas the PEX6 mutantwere unable to localize both fluorescent proteins. Deletion of PEX5 and PEX6 triggered reduced growth on long chain fatty acids and butyrate, while thePEX7 deletion mutants utilized fatty acids other than butyrate. Virulence on wheat heads was greatly reduced in the PEX5 and PEX6 deletion mutants,because they were impaired in spreading from inoculated florets to the adjacent spikelets through rachis. Disruption of PEX5 and PEX6 droppedsurvivability of aged cells in planta and in vitro due to the accumulation of ROS followed by necrotic cell death. We suggest that PTS1-type peroxisomalcatalases are responsible for ROS scavenging. These results demonstrate the functions of peroxisomes in survival and ROS detoxification of filamentousfungi.196. roGFP and anti-oxidant defences in the rice blast fungus Magnaporthe oryzae. Marketa Samalova, Sarah Gurr, Mark Fricker. Plant Sciences,University of Oxford, Oxford, United Kingdom.The ascomycete fungus Magnaporthe oryzae causes rice blast disease. Germination and development of its infection structure, the appressorium on thehost surface is orchestrated by a complex set of signals from within the fungus, and later between the fungus and resistant or susceptible plant that,respectively, either triggers host defence or leads to infection. Host defences involve localised production of reactive oxygen species (ROS), which eitherkill the pathogen directly or block fungal invasion by oxidative cross-linking of cell wall glycoproteins. By contrast, infection suggests that the invadingfungus can tolerate or, indeed, bypass such defences. Here, we report rice blast fungus’ capacity to withstand transient oxidative stress during earlydevelopment. We determine the intrinsic cytoplasmic cell glutathione (GSH) concentration by confocal imaging of monochlorobimane, which becomesfluorescent when conjugated to GSH. The redox poise of the glutathione pool was measured by 4-D confocal excitation ratio imaging of GRX1-roGFP2. Wereveal that this fungus has an extraordinary ability to tolerate severe insults of H 2O 2, with rapid recovery of its reduced GSH pool and thence continued168